The human genome project has inspired significant advancements in high-throughput technologies for the study of function and activation in gene expression. However, in order to truly tackle disease, the changes associated with gene expression at the mRNA and proteome levels has to be understood. Since the post-genomic era, the field of proteomics-the systematic profiling of proteins under a given set of conditions such as diseased versus healthy state- has grown tremendously. One of the major problems of proteomics is the low abundance of post-trans la tiona I ly modified proteins in complex biological mixtures such as phosphoproteins. This mandates a need for robust and highly sensitive detection system in which mass spectrometry (MS) has evolved as the method of choice for these analyses due to its fast experimental timescales and sensitivity. The long term goal of this proposal is to develop an ultra-sensitive ion trap mass spectrometry technique to routinely carry out comprehensive MS/MS analysis of complex biological mixtures. The key to this development is the ability to monitor all ions that are stored in an ion trap without discarding any, which is typically done in a standard proteomics experiment. The specific aims of this proposal are to: (1) Optimize the recently constructed high-capacity ion trap instrument by isolating the pressure within the ion trap to allow us to use optimal gases in the various regions of the instrument: helium in the high-capacity ion trap, N2 in the ion source cooling region, and N2 or argon in the collision cell. In addition, we will modify the geometry of the high-capacity ion trap to increase the resolution of the instrument. These straightforward improvements are expected to further enhance the performance of the high-capacity ion trap instrument. (2) The next phase of the research will couple an orthogonal injection reflectron TOP mass analyzer to the high-capacity ion trap. (3) Finally, future experiments will be geared towards the comprehensve sample analysis using this new high-capacity ion trap TOP MS instrument. Abnormal levels of protein phosphorylation are known to be associated with major diseases such as cancer, diabetes, and rheumatoid arthritis. The ability to identify and study these phosphorylation processes will ultimately bring us a step closer to finding a cure.